Combined use of triglycerides containing medium chain fatty acids and exogenous lipolytic enzymes as feed supplements

ABSTRACT

The present invention provides a feed composition comprising about 0.01% to about 20% by weight of triglycerides and about  1  ppm to about 10,000 ppm of exogenous active lipolytic enzyme, wherein the triglycerides contain about 50% to about 100% of C 6 -C 10  medium chain fatty acids. The present invention also provides methods of using the feed composition of the present invention for controlling lipolysis, as well as treating growth impairment and digestive upsets in an animal.

This application is a continuation-in-part application of U.S. patent application Ser. No. 10/009,235, filed Jun. 11, 2002, which is a 35 U.S.C. §371 national phase application of PCT/EP00/05192, filed Jun. 6, 2000, which claims the benefit of European patent application No. 99870120.5, filed Jun. 7, 1999, the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention generally relates to animal feeds. In particular, the present invention relates to the use of triglycerides containing medium chain fatty acids combined with exogenous active lipolytic enzymes as feed supplements for animals.

BACKGROUND OF THE INVENTION

Early weaning (3 to 4 weeks of age) of piglets has become a general practice in pig husbandry systems for increasing the productivity and maintaining the profitability. Early weaning, however, burdens the piglet with a lot of stresses, mainly of environmental, nutritional and immunological origin, combined with a more or less pronounced depression of feed intake and mobilization of body reserves. Mal-digestion and mal-absorption often aggravate the situation resulting in digestive upsets due to bacterial overgrowth and/or viral infections. These phenomena greatly interfere with the profitability of the enterprise. These issues have been discussed in a vast body of literatures, e.g., VAN DER PEET (1992); and PARTRIDGE (1993).

Current solutions aim at adapting the feed to the digestive capacity of the piglet by improving the acceptability of the feed through the use of specific ingredients (e.g., milk powder and derivates, such as whey, lactose, dried blood serum, and flavors), with or without an increase of the energy content of the feed. An increase of the energy content can be obtained among others by including easily digestible or metabolizable fats, for example, medium chain triglycerides (MCTG). MCTG in this context have been well documented both in neonatal (ODLE, 1999) and in weaned piglets (CERA et al., 1989), and their utility in this aspect is determined by their specific digestive and metabolic fate (BACH & BABAYAN, 1982).

Digestive upsets are prevented and/or treated by supplementing the feed with antimicrobial substances (e.g., antibiotics or chemotherapeutics, referred to as “antibiotics” hereafter). The effects of the above interventions (prevention or treatment) mostly result in a pronounced improvement of the growth performances (referred to as “growth promotion” hereafter). This growth promotion is mainly due to, depending on the circumstances, an improved feed intake combined with a better feed conversion (i.e., kg feed/kg gain). However, there is a growing concern about the use of antibiotics for growth promotion in animal production systems; in particular, there is a well-considered fear for the risk of the emergence of cross-resistance to some last-resort antibiotics used in human medicine (CORPET, 1996; WEGENER et al., 1998). Therefore, most of those antibiotics (or so called “growth promoters”) are already or will be banned in the near future in the European Union, which justifies an urgent need for alternatives.

Because there is a general belief that digestive pathology in early weaned pig is mainly caused by Gram-negative bacteria (especially E. coli) and that Gram-positive lactic acid bacteria (e.g., Bifidobacteria, Lactobacilli) have a protective and/or antagonistic effect, the currently proposed alternatives are selected for their anti-E. coli activity. Examples of such alternatives include cupper and zinc compounds, selected organic acids such as short chain fatty acids (SCFA) (e.g., formic, acetic and propionic acids), lactic, fumaric, citric, malic and sorbic acids, probiotics (mainly lactic acid bacteria) and/or prebiotics (mainly bifidogenic oligosaccharides, or so called NDO's). Cupper and zinc compounds are effective but are not acceptable because they pollute the environment, and results obtained with probiotics and/or prebiotics are usually unpredictable and generally disappointing (CHESSON, 1994).

Similar problems exist in other animal species as well as in animals of other age groups. Only SCFA and the “classical” organic acids are the most promising alternatives at this time (ROTH et al., 1998). However, they are needed in rather high doses to be effective. That is, the utility of SCFA and “classical” organic acids is limited as there involves a high cost, a corrosive nature and an averse taste which interferes greatly with the feed intake of the animals.

The antimicrobial effects of fatty acids (FA) in general and their salts (e.g., soaps) are already known for decades. A re-evaluation of the antimicrobial effects of selected FA and derivates thereof was given in the review of KABARA (1978), wherein special attention was given to lauric acid (C₁₂, a member of the MCFA family) and derivatives.

Relatively important contribution of MCFA has been documented in the milk-lipid of certain animal species (e.g. rabbit, goat, horse), while low or zero concentration of MCFA in sow's milk renders limited contribution (DIERICK, 1998). In most mammals, there is a more or less pronounced preduodenal (i.e., not of pancreatic origin) lipolytic activity originating from lingual or gastric secretions. The activity of those lipases is independent of the presence of colipase and bile acids, is active and stable in a broad range of pH's, and has a preference for MCFA in milk fat. The preduodenal lipase activity is high in preruminant calves and rabbits, moderate in piglets and absent in poultry (MOREAU et al., 1988). However, an excess of MCFA can have important side-effects, for example, it can be hypnotic in new born pigs (ODLE, 1999), and is a strong stimulus for cholecystokinin (CCK, an intestinal hormone with a pronounced satiating activity which could interfere with the feed intake) (LEPINE et al., 1989). In addition, a lower feed intake could also due to the strong odor (goat-like) and averse taste of free MCFA, although data in this context are scarce and non-conclusive.

The mechanism by which SCFA, MCFA and other organic acids excert antimicrobial activities has been well documented in the literature. The belief is that undissociated or non-ionized acids (RCOOH) are lipid-permeable and can pass across the microbial cell membrane and subsequently dissociate (RCOOH→RCOO⁻+H⁺) in the more alkaline interior of the microorganism. This dissociation or ionization reaction results in an acidification of the intracellular pH, which would be below permissible levels for microbes to survive. In other words, organic acids act as protonophores that increase the inward leak of H⁺ so that efflux is not rapid enough to alkalinize the cytoplasm again. Physicochemical characteristics of the organic acids greatly influence their ability to act as protonophores, for example, molecular weight, pKa (dissociation constant) and solubility. In addition, physiological environment in which the organic acids are present, especially the pH in the different locations of the gastrointestinal tract, is also a very important factor. Furthermore, the type of the microbial envelope, i.e., mainly peptidoglycan in Gram-positive bacteria and lipopolysaccharide in Gram-negative bacteria, greatly influences the passage of the organic acids through the membrane as well.

The specific characteristics of MCTG being a readily available energy source have also been well documented. Their beneficial effects as an energy source are summarized in BACH & BABAYAN (1982): (1) MCTG are digested, absorbed and transported rapidly in disorders where digestion and absorption are not optimal. Such disorders of mal-digestion and mal-absorption are frequently observed in newly weaned piglets, and are attributed to a sharp drop in the activity of most of the digestive enzymes. For example, the deficiency of lipolytic enzymes shortly after weaning is very pronounced; (2) MCTG are oxidized rapidly in the organism and are a source of abundant and rapidly available energy. However, high doses of MCTG are ketogenic and can have narcotic side effects, which are certainly undesirable in piglets.

Furthermore, the depressive effect on the voluntary feed intake, by activation of CCK, is unwanted. Also, the strong unpleasant odor of the free MCFA which evaporates relatively easily is also unwanted either by the producer or the animals.

Thus, there is a long-standing need for a feed supplement for animals, especially for early weaned pigs in order to prevent and/or alleviate the existing problems. The present invention fulfills this need.

SUMMARY OF THE INVENTION

The present invention is related to a feed supplement or feed composition and its use for animal feeds, especially for early weaned pigs in order to prevent and/or alleviate existing feeding problems. Such feed supplement or feed composition can be used as part (mostly 1 to 5%) of a complete feed (or often referred to as “feed formula”).

In particular, the present invention is directed to a feed composition comprising about 0.01% to about 20% by weight of triglycerides and about 1 ppm to about 10,000 ppm of exogenous active lipolytic enzyme, wherein the triglycerides contain about 50% to about 100% of C₆-C₁₀ medium chain fatty acids (MCFA).

The present invention is also directed to a method for controlling lipolysis in the gastro-intestinal tract of an animal. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.

The present invention is further directed to a method for treating growth impairment of an animal. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.

The present invention is still further directed to a method for treating digestive upsets of an animal. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.

The foregoing and other advantages of the present invention will be apparent to one of ordinary skill in the art, in view of the following detailed description of the preferred embodiment of the present invention, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrate the in vitro release of medium chain fatty acids (MCFA) (expressed as g/100 g of triglycerides (TG)) at different pH's from four different TG tested (FIG. 1A: coconut oil; FIG. 1B: MCTG1; FIG. 1C: MCTG2; FIG. 1D: butterfat) with different lipolytic enzymes selected. The lipolytic enzymes, coded L1 to L6, were used in a dose of 10,000 ppm on the basis of TG. The release of MCFA was studied in a buffered medium at pH 2, 3, 4 or 5 as these pH's being representative for the pH conditions prevailing in vivo in the stomach.

FIGS. 2A-2C illustrate the total and individual bacterial counts (expressed as log₁₀ Colony Forming Units (CFU) per g fresh contents) in the stomach contents of cannulated pigs fed with 5% coconut oil, MCTG1 and butterfat, respectively. The first group of bars in each figure represents the results obtained without lipolytic enzymes, while the second and third groups illustrate the results obtained with the addition of lipolytic enzymes L2 and L5 (1,000 ppm on feed basis), respectively. The first bar of each group represents the total count; whereas the second to fourth bars represent the counts of lactobacilli, streptococci and E. coli, respectively.

FIG. 3 illustrates the fat fractions in the gastric contents (expressed as g/100 g contents) of the cannulated pigs fed with 5% coconut oil (V1-V3), MCTG1 (V4-V6) or butterfat (V7-V9). The proportion of free fatty acids (FFA) to total fatty acids (FA) is given for the feeds with the different TG used without lypolytic enzyme (V1: coconut oil, V4: MCTG1; V7: butterfat) or with the supplementation of lypolytic enzymes L2 or L5 (1,000 ppm on feed basis).

FIGS. 4-14 each is a graph plotting the results of the growth inhibition of Salmonella Thypimurium using different compositions according to the embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention aims at providing new feed supplements for animal feeds, particularly for early weaned pigs, which can replace the commonly used yet contested antibiotics and other growth enhancers.

The present invention is related to a feed supplement or feed composition that is a premix of feed additives (vitamins, minerals, antibiotics, among others) with a carrier for use as part (mostly 1 to 5%) of a complete feed (or often referred to as “feed formula”). The feed composition comprises a list of different feed ingredients used in the complete feed (or feed formula).

In particular, the present invention provides a feed composition comprising at least one triglyceride (TG) containing medium chain fatty acids (MCFA) (C₄-C₁₂, preferably C₆-C₁₀ MCFA) combined with at least one exogenous active lipolytic enzyme (e.g., esterase and/or lipase) as well as its use as a feed supplement for animal feeds, especially for early weaned pigs in order to prevent and/or alleviate existing feeding problems at this time. The addition of the combination of TG and exogenous active lipolytic enzymes to an animal feed, which produces a feed formula, surprisingly results in a physiological environment in the stomach which regulates and stabilizes the gastrointestinal microflora. This effect, combined with the fact that an easily digestible and metabolizable source of energy is provided by MCFA, further results in a marked improvement of the growth which is comparable with the growth promotion obtained with commonly used (yet contested) antibiotics and other growth enhancers without negative side effects to the animal, the feed industry and the consumer.

In one embodiment of the present invention, there is provided a feed composition comprising about 0.01% to about 20% by weight of triglycerides and about 1 ppm to about 10,000 ppm of exogenous active lipolytic enzyme, wherein the triglycerides contain about 50% to about 100% of C₆-C₁₀ medium chain fatty acids (MCFA).

MCFA according to the present invention include both even and odd fatty acids, such as fatty acids containing C₆ (e.g., caproic acid (or hexanoic acid)), C₇ (e.g., heptanoic acid), C₈ (e.g., caprylic acid (or octanoic acid)), C₉ (e.g., pelargonic acid), and/or C₁₀ (e.g., capric acid (or decanoic acid)).

In a preferred embodiment, the triglycerides according to the present invention, also referred as medium chain triglycerides or MCTG, contain about 60% to about 100%, preferably about 60% to about 100%, preferably about 70% to about 100%, more preferably about 80% to about 100%, more preferably about 90% to about 100%, or yet more preferably about 100% of C₆-C₁₀ MCFA. MCFA can be caproic acid, heptanoic acid, caprylic acid, pelargonic acid, capric acid, or combination thereof. MCTG can be caproic/caprylic/capric triglycerides, heptanoic/pelargonic triglycerides, pelargonic triglycerides, heptanoic triglycerides, caprylic/capric triglycerides, caproic/caprylic triglycerides, caproic/capric triglycerides, caproic triglycerides, caprylic triglycerides, capric triglycerides, or combination thereof. Preferably, MCTG can be caproic/caprylic/capric triglycerides, caprylic/capric triglycerides, caproic/caprylic triglycerides, caproic/capric triglycerides, caproic triglycerides, caprylic triglycerides, capric triglycerides, or combination thereof.

As used herein, the term “medium chain triglyceride (MCTG)” shall refer to a glyceride in which the glycerol is esterified with three medium chain fatty acids (MCFA). As used herein, the term “caproic triglyceride” or “glyceryl tricaproate” shall refer to a glyceride in which the glycerol is esterified with three caproic acids. As used herein, the term “caprylic triglyceride” or “glyceryl tricaprylate” shall refer to a glyceride in which the glycerol is esterified with three caprylic acids. As used herein, the term “capric triglyceride” or “glyceryl tricaprate” shall refer to a glyceride in which the glycerol is esterified with three capric acids.

As used herein, the term “caproic/caprylic/capric triglyceride” shall encompass a triglyceride wherein the glycerol is esterified with caproic, caprylic and/or capric fatty acids (also termed “glyceryl caproate caprylate caprate”), as well as a combination of glyceryl tricaproate, glyceryl tricaprylate, glyceryl tricaprate, glyceryl caprylate caprate, glyceryl caprylate caproate, glyceryl caproate caprate, and glyceryl caproate caprylate caprate.

As used herein, the term “caprylic/capric triglyceride” shall encompass the triglyceride wherein the glycerol is esterified with caprylic and capric fatty acids (also termed “glyceryl caprylate caprate”, as well as a combination of glyceryl tricaprylate, glyceryl tricaprate, and glycerol caprylate caprate.

As used herein, the term “caproic/caprylic triglyceride” shall encompass the triglyceride wherein the glycerol is esterified with caproic and caprylic fatty acids (also termed “glyceryl caproate caprylate”), as well as a combination of glyceryl tricaprylate, glyceryl tricaproate, and glyceryl caprylate caproate.

As used herein, the term “caproic/capric triglyceride” shall encompass the triglyceride wherein the glycerol is esterified with caproic and capric fatty acids (also termed “glyceryl caproate caprate”), as well as a combination of glyceryl tricaprate, glyceryl tricaproate, and glyceryl caprate caproate.

In a more preferred embodiment, the C₆-C₁₀ MCFA are C₆ MCFA, C₈ MCFA, C₁₀ MCFA, or combination thereof. In this case, MCTG according to the present invention contain about 0% to about 100% of C₆ MCFA, about 0% to about 100% of C₈ MCFA and about 0% to about 100% of C₁₀ MCFA, wherein the sum of all three fatty acids is about 50% to about 100%, preferably about 60% to about 100%, preferably about 70% to about 100%, more preferably about 80% to about 100%, more preferably about 90% to about 100%, yet more preferably about 100%.

The above concentration of MCTG can range from, for example, 0.0125% to about 20%, from about 0.015% to about 20%, from about 0.02% to about 20%, from about 0.04% to about 18%, from about 0.05% to about 15%, from about 0.08% to about 15%, from about 0.1% to about 15%, from about 0.5% to about 15%, from about 0.8% to about 15%, from about 1% to about 15%, or from about 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% to about 20%.

MCTG according to the present invention may be a naturally occurring triglyceride-containing composition. Examples include but are not limited to coconut oil, palm kernel oil, babassu oil, cohune oil, tacum oil, cuphea oil derived from plant seeds, milk of mammalian species (e.g., milk from horse, rat, goat and rabbit), and butterfat. Alternatively, MCTG may comprise one or more industrially prepared triglycerides or a mixture of naturally occurring and industrially prepared triglycerides. Industrially prepared triglycerides may be produced by esterification, such as trans- or inter-esterification of C₄-C₁₂, preferably C₆-C₁₀ fatty acids. Examples of commercial sources of chemically synthesized MCTG include but are not limited to those given in Table 10 or those exemplified in the Materials section of the Examples.

In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caproic triglycerides, and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.

In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caprylic triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.

In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of capric triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.

In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caproic/caprylic triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.

In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caprylic/capric triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.

In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caproic/capric triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.

In a preferred embodiment, the present invention provides a feed composition comprising about 0.01% to about 20% by weight of caproic/caprylic/capric triglycerides and about 1 ppm to about 10000 ppm of exogenous active lipolytic enzyme.

In a preferred embodiment, the lipolytic enzyme component is added in a concentration of about 1 ppm to about 10,000 ppm to the feed composition, for example, about 1 ppm to about 50 ppm, about 10 ppm to about 10,000 ppm, about 50 ppm to about 10,000 ppm, about 100 ppm to about 10,000 ppm, about 200 ppm to about 10,000 ppm, about 250 ppm to about 10,000 ppm, about 300 ppm to about 10,000 ppm, about 500 ppm to about 10,000 ppm, about 800 ppm to about 10,000 ppm, about 900 ppm to about 10,000 ppm, or about 1,000 ppm to about 10,000 ppm. Preferably, the lipolytic enzyme is added to the feed composition in a concentration of about 10-80 mg/g of MCTG. For example, the exogenous active lipolytic enzymes are added in a concentration of about 10 mg/g, 15 mg/g, 20 mg/g, 25 mg/g, 30 mg/g, 35 mg/g, 40 mg/g, 45 mg/g, 50 mg/g, 60 mg/g, 70 mg/g or 80 mg/g of MCTG.

The lipolytic enzyme component according to the present invention may comprise a lipase or an esterase, a mixture of lipases and/or esterases. Such lipases or esterases may be naturally occurring or industrially prepared, and may be from a microbial, mammalian or plant origin.

Examples of commercially available plant lipases include but are not limited to lipases from wheat, castor bean, rape, mustard and lupin. Examples of commercially available microbial lipases include but are not limited to the lipases as given in Table 11 or those exemplified in the Materials section of the Examples.

Examples of commercially available esterases include but are not limited to pregastric esterase (PGE) from sublingual tissue of calf, kid and lamb, rennet paste from engorged abomasa of calf, kid and lamb, esterase from rabbit liver or porcine liver.

In still a preferred embodiment, the feed composition further comprises an animal feed comprising carbohydrate, protein, saccharides and a vitamin-mineral premix, for example, an animal feed comprising starch, dextrose, casein and a vitamin-mineral premix. In still another preferred embodiment, the feed composition further comprising an animal feed comprising maize, barley, wheat, dried acid whey, cassaya, herring meal, soybean, soy-flour and a vitamin-mineral premix.

A feed formula comprising 1 to 5% of the feed composition of the present invention is also provided.

The feed composition or feed formula according to the present invention is useful for feeding animals, particularly pigs, cattle, horse, sheep, rabbits and poultry, and more particularly early weaned animals. It is also particularly useful as a growth promoter, an antimicrobial agent as well as for preventing digestive upsets.

Although the feed composition or feed formula is particularly useful for early weaned animals such as piglets, it is not excluded from being used as a feed supplement for pigs of other age categories or as a feed supplement or feed formula for other species of animals.

In another embodiment of the present invention, there is provided a method for preparing a feed composition or feed formula by mixing together different MCTG and lipolytic enzyme components according to the present invention.

In still another embodiment of the present invention, there is provided a method for controlling lipolysis in the gastro-intestinal tract of an animal, e.g., an early weaned piglet. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.

In yet another embodiment of the present invention, there is provided a method for treating growth impairment of an animal, e.g., an early weaned piglet. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.

In still yet another embodiment of the present invention, there is provided a method for treating digestive upsets of an animal, e.g., an early weaned piglet. This method includes the step of administering to the animal a therapeutically effective amount of the feed composition according to the present invention.

In the present study, a broad range of organic acids (SCFA, MCFA and other commonly used organic acids in the feed and food industry) were first tested in vitro for their antibacterial activity against the dominant bacteria of the small intestinal microflora. Unexpectedly, SCFA and the commonly used organic acids were found to be bacteriostatic only at higher concentrations (0.02 to 0.04 M) for the Gram-negative flora (and to a lesser extent for Streptococci). However, MCFA were shown to have high bacteriostatic and bactericidal activity against both Gram-positive and Gram-negative bacteria. The antibacterial activity of MCFA was inversely related to pH, wherein MCFA had a higher antibacterial activity at a lower pH. For example, a relatively high proportion of MCFA was in the undissociated form.

In the same in vitro study, a temptative minimal bactericidal concentration of 0.005 to 0.01 M was tested. Also unexpectedly, the antibacterial spectrum of the antibiotic growth promoters used in the intensive animal production could be duplicated completely when a combination of MCFA was used.

In order to maximize the positive effects and minimize the negative characteristics of MCFA, a combination of a MCFA-containing triglyceride and a lipolytic enzyme is used as a feed supplement in the present invention, with the intention that sufficient MCFA should be released in the stomach to have a sterilizing effect, which would result in a lesser bacterial load in the intestinal tract thereby preventing digestive upsets. This sterilizing effect, combined with the characteristics of MCFA being a readily available energy source and the supplementation of the natural lipase activity in the stomach and upper intestine by the exogenous active lipolytic enzyme(s), was shown to have unexpectedly resulted in a growth promotion without the use of antibiotics. It appears that the gradual release and absorption of the free MCFA unexpectedly avoided the unwanted side effects of MCFA.

The following examples and drawings merely serve to illustrate the present invention and are not meant to be limiting in any manner.

EXAMPLES Materials

Two naturally occurring substances which are rich in medium chain fatty acid-containing triglycerides, i.e., butterfat and coconut oil, and two commercially available sources of MCTG, i.e., MCTG1 (Aldo MCT Kosher Food Grade, commercialized by LONZA Inc. (Fair Lawn, N.J. 070410, USA)) and MCTG2 (Stabilox-860, commercialized by LODERS-CROKLAAN BV (NL-1521 AZ Wormerveer)) were used in the present study. Also, lipolytic enzymes L1 (Lipozyme 10.000L, NOVO Nordisk A/S, 2880 Bagsvaerd, Denmark); L2 (Lipase 10.000P, Biocatalysts Ltd., CF37 5UT Pontypridd, Wales, UK); L3 (TP 516P, Biocatalysts Ltd., CF37 5UT Pontypridd, Wales, UK); L4 (LIPOMOD 224P, Biocatalysts Ltd., CF37 5UT Pontypridd, Wales, UK); L5 (Lipase SAIKEN, NAGASE & Co, Chuo-ku, 103 Tokyo, Japan); and L6 (Lipase ITALASE C, SBI, Systems Bio-Industries, Inc., WI 53187-1609 Waukesha, USA) were used in the present study.

The selection of above triglycerides and lipolytic enzymes does not exclude potential usefulness of other triglycerides and lipolytic enzymes and combinations thereof for the purposes of the present invention.

Methods for Extraction and Analysis of Different Lipid Compounds

A lipid extraction procedure was used, wherein hexane/iso-propanol (3/2, v/v) was used to avoid any solvent evaporation thereby preventing any loss of MCFA due to their great volatility.

Next, H₂SO₄ was used to catalyze esterification of MCFA in the same extraction medium, wherein isopropyl esters (FAIPE) was formed without loss of shorter esters or alteration of polyunsaturated higher MCFA. FAIPE appeared in the upper hexane phase.

For the calculation of the concentration, quantitative capillary column (DB-225, 30 m, ID 0.25 mm, Film 0.25 μm) GLC chromatography of individual FAIPE was conducted, wherein two internal standards were used, i.e., C₉ for C₄-C₁₂ and C₁₇ for C₁₄ to C_(18:3). Coefficients of variation on the response factors amounted to 0.94% for C₉ and 2.51% for C₁₇.

Individual free fatty acids were extracted using the above-described lipid extraction procedure with a strong anion exchange resin Amberlyst 26 before esterification in the same medium, and then analyzed by capillary GLC mean recovery of added free fatty acids amounted to 101.9%.

Example 1 In Vitro Screening of MCTG and Lipolytic Enzymes for Lipolysis at Different pH's Simulating Gastric Conditions

Lipolytic enzymes L1-L6 were selected based on their commercial availability and cost. MCTG were selected based on their specific MCFA content in the fat as specified in Table 1 below.

TABLE 1 MCFA Concentration (g/100 g FA) in Selected Triglycerides C₄ C₆ C₈ C₁₀ C₁₂ Butterfat 3.4 2.1 1.2 2.6 3.0 Coconut oil 0 0.7 8.5 6.2 48.8 MCTG 1 0 2.8 69.1 27.7 0.4 MCTG 2 0 0.2 57.5 42.3 0.0

The in vitro incubations were done for 180 min at 37° C. in a shaking water bath in buffered circumstances at different pH's: a glycine buffer was used for incubations at pH 2 and pH 3; and an acetate buffer was used for incubations at pH 4 and pH 5. The parameters used for the incubations were chosen in order to simulate as closely as possible the in vivo conditions in gastric contents. The medium used for the incubations was made up of the following ingredients: 0.250 g of the selected TG+2.250 g of a synthetic feed (based on starch, dextrose, casein and a vitamin-mineral premix)+10 ml buffer solution+0.5 ml pepsine solution (50 mg in 100 ml aqua dest)+10,000 mg/kg fat (i.e., 10,000 ppm) of the selected commercial lipolytic enzyme preparation. The fat could be molten when needed; otherwise there were no special preparations (e.g., dispersion or emulgation) of the fat.

The results of the incubations are demonstrated in FIGS. 1A-1D), which represent the released MCFA in g/100 g of TG for the different TG tested. The hydrolytic activity was the highest at pH 2 to 5 with each of the enzymes, which matches the pH normally occurring in vivo in the stomach of pigs. The amount of released free MCFA seems to be dependent on the amount present in the original source of TG. The amount of MCFA released using L2 was about 3.7% with coconut oil, about 18.2% with MCTG1, about 15% with MCTG2, and about 0.65% with butterfat.

Example 2 In Vivo Experiment with Gastric Cannulated Pigs for Study of In Situ Release of MCFA by Endogenous and Exogenous Lipolytic Enzymes

Three female pigs (Belgian Landrace and stress free) with an initial weight of about 8.5 kg were prepared with a gastric cannula using the technique of DECUYPERE et al. (1977). The cannulae were placed midway the curvatura major in the fundic region.

Three TG (coconut oil, MCTG1 and butterfat) and two lipases (L2 and L5) were selected for this experiment. Nine feeds were prepared using 95% of a commercial feed for piglets and 5% of the selected (eventually molten) TG with or with supplementation of the selected lipases (see Table 2 for the V codes used hereafter). The fats were simply poured on the meal and thoroughly mixed in a horizontal mixer. The concentration of the lipases was 1,000 ppm of the commercial preparation in the feed.

TABLE 2 Summary of Feeds Coconut oil: V1: 95% piglet feed + 5% coconut oil V2: idem + 1,000 ppm L2 V3: idem + 1,000 ppm L5 MCTG1: V4: 95% piglet feed + 5% MCTG1 V5: idem + 1,000 ppm L2 V6: idem + 1,000 ppm L5 Butterfat: V7: 95% piglet feed + 5% butterfat V8: idem + 1,000 ppm L2 V9: idem + 1,000 ppm L5

The composition of the piglet feed was based on maize, barley, dried acid whey, cassaye, herringmeal, and soybean oil, and was supplemented with a vitamin-mineral premix. The feed contained no growth promoting supplements. The proximate analysis of the feeds (V1, V4 and V7) in % of as given was: DM: 90.6, 90.7 and 90.8; total ash: 7.8, 7.9 and 8.5; crude protein: 15.1, 15.4 and 14.8; crude fat: 8.5, 8.3 and 8.3.

The feed was given dry in three equal meals (9, 13 and 17 h) at 85% of the ad libitum intake of pigs with a comparable weight. The experiment had a 3×3 Latin square design. There were no health problems or feed refusals. Statistics were done using ANOVA (1997), differences were at p<0.01 to p<0.05 (**) or p<0.1 (*).

Sampling of the gastric contents for the chemical analysis was done on 2 consecutive days, 2 times a day, and 30 min after the 9 h and 13 h meal. The pH was measured directly; thereafter the samples were stored at −20° C. till further analysis.

The sampling of the gastric contents for the bacteriological analysis was done during 1 day, and 90 min after the 9 and 13 h meal. The bacterial counts were done using the technique of VAN DER HEYDE et al. (1964). The media (all from OXOID, UK) used were RCM agar+hemin for the total count (48 h, anaerobic), Rogosa agar for the Lactobacilli (48 h, anaerobic), Slanetz & Bartley agar for the fecal Streptococci (24 h, aerobic), and EMB agar for E. coli (24 h, aerobic). All incubations were performed at 37° C. Results are expressed as log₁₀ colony forming units (CFU)/g fresh contents.

It is shown that the pH of the stomach contents measured 30 and 90 min after feeding did not differ between the treatments (feeds) and ranged between 4.2 and 5.01. This is within the optimum range for the lipolytic activity of L2 and L5 as was found in the above experiment described in Example 1. The results of the bacteriological counts are presented in Table 3 and in FIGS. 2A-2C, which suggest that with each TG, the enzymes cause a reduction of the total count and individual lactobacilli count.

TABLE 3 Bacteriological Counts in Gastric Contents of Piglets (log₁₀ CFU/g fresh contents: mean ± s.d) (n = 6) Total Lacto. Strepto E. coli Coconut oil V1 6.4 ± 0.8  6.0 ± 0.8 4.3 ± 1.0  2.3 ± 1.2 V2 5.2 ± 0.3**  5.0 ± 0.3** 4.1 ± 0.6  2.4 ± 1.4 V3 5.3 ± 0.6**  4.9 ± 0.7** 2.7 ± 1.6* 2.6 ± 2.1 MCTG1 V4 6.1 ± 0.2  5.7 ± 0.5 5.2 ± 0.4  2.9 ± 1.6 V5 4.2 ± 0.5**  3.7 ± 0.5** 0.0**  1.0 ± 1.5** V6 3.4 ± 1.7**  2.7 ± 1.4**  0.5 ± 1.2**  0.5 ± 1.2** Butterfat V7 6.4 ± 0.4  5.7 ± 0.8 5.0 ± 0.6  4.1 ± 0.5 V8 5.6 ± 0.9*  5.5 ± 0.3 4.0 ± 0.7*  3.4 ± 0.1* V9 5.7 ± 0.5*  5.5 ± 0.5 4.0 ± 0.7* 4.5 ± 1.4 *, **denote differences per TG within the column

The data show the following: (1) with coconut oil, both L2 and L5 reduced 10-fold of the total count and lactobacilli count; (2) with MCTG1, both enzymes had a very pronounced (mostly p<0.001) effect and reduced the total count and individual lactobacilli count by a factor of 100 to 1,000, and reduced individual streptococci and E. coli counts to non detectable levels; and (3) with butterfat, there was a 10-fold reduction of the total count and individual streptococci count.

It is concluded that the combination of a MCTG and a lipolytic enzyme in the feed is able to suppress the total bacterial count and individual counts of the dominant flora. This effect most likely is due to the release of free MCFA from the triglycerides used, which was confirmed by the chemical analysis of the different fat fractions in the gastric contents collected during the present study. The results of the chemical analysis are given in FIG. 3, wherein the amounts of total and free fatty acids (total FA and FFA) per 100 g of fresh gastric contents are presented. Also, the results expressed as g of FFA per 100 g of total FA in the stomach contents, or the degree (%) of hydrolysis of the triglycerides, are given in Table 4.

TABLE 4 Degree of Hydrolysis* of Different Triglycerides Used as Influenced by L2 or L5 Control +L2 +L5 Coconut oil V1 V2 V3 16.5 43.2 44.8 MCTG1 V4 V5 V6 18.9 58.5 60.9 Butterfat V7 V8 V9 16.8 46.8 45.8 *g FFA/100 g of total FA in fresh gastric contents

There was no preferential release of specific FA (results not shown), that is, the release of individual FA is roughly proportional to their content in the triglycerides used. Results presented in FIG. 3 and Table 4 suggest that the endogenous lipolytic activity in the stomach of the piglets hydrolyses about 16-19% of the triglycerides. The addition of the exogenous lipolytic enzymes increases the hydrolysis about 3-fold. FFA released without exogenous enzymes result from the activity of the endogenous preduodenal lipases. These results suggest that the exogenous lipolytic enzymes used greatly enhanced the release of FFA from each TG tested.

It is striking and unexpected that the release of MCFA runs parallel with the degree of suppression of the bacterial load in the stomach with the most efficient suppression observed with the combination MCTG1+L5, which caused 60.9% hydrolysis of the triglycerides in the stomach (corresponding with a concentration of about 1% of FFA and 0.6% of MCFA), followed by coconut oil+L5 (0.8% FFA and 0.3% MCFA) and butterfat+L5 (0.8% FFA and 0.06% MCFA).

Example 3 Zootechnical Experiment in Commercial Settings: Growth Performance Combined with Ex Vivo Observations on the Gastric Contents

This experiment was designed to check if the above-mentioned concept was applicable and suitable in commercial settings and if obtained, whether a growth promotion was comparable with the growth promotion obtained in early weaned piglets with antibiotics or a combination of commonly used organic acids with proven effectiveness.

244 freshly weaned piglets (Seghers Hybrid F1, initial weight about 6.5 kg) were divided according to litter, sex and weight in 4 groups: A: 68; B: 61; C: 60 and D: 55 piglets. The experiment was run in commercial settings in temperature controlled facilities.

The feed composition used was based on barley, wheat, maize flakes, extruded maize, extruded soybeans, soy-flour, herring meal, 2.5% TG, and a commercial premix (mainly based on milk products, vitamins+minerals) for early weaned piglets (12.5%). The treatments (A to D) differed in TG and the additives used (see Table 5). The feeds contained no growth promoting antibiotics. Feed A was a negative control, feed D a positive control containing a mix of commonly used organic acids. The calculated proximate analysis of the feeds used was equalized. The formulated contents were (% fresh): DM: 90.0 to 88.8, crude protein: 18.7 to 18.9, crude fat 6.9, total ash: 5.1-5.3. The energy content was (Nef97): 2463-2475 kcal/kg, the ileal digestible amino acids were set at: Lys: 1.07%, Met+Cys: 0.65, Thr: 0.66, Try 0.19.

TABLE 5 Treatments Used in Zootechnical Experiment Treatment A B C D TG (2.5%) soybean oil MCTG2* MCTG2 soybean oil Lipase (L5) — — 1000 ppm** — Supplement — — — 1.5% organic acids*** *MCTG2 was selected upon commercial availability **based on fresh feed ***0.25% citric acid + 0.75% fumaric acid, 0.5% Na-formiate (as specified by the feed manufacturer)

The feed was prepared by a commercial feed company which used a spray-equipment for fats and other liquid supplements. The feed was offered dry, ad libitum; water was continuously available via a nipple.

The experiment lasted 3 weeks. The piglets were weighed individually at the start of the experiment and weekly thereafter; feed intake was recorded daily per two pens (joint feed hopper for two pens with about 15 piglets each). Therefore statistics only could be done for the weights. The visual health condition of the pigs per pen was checked daily and coded on a scale from 0 (extremely bad) to 10 (excellent). The zootechnical results on a weekly basis are presented in Table 6.

TABLE 6 Zootechnical Performances of Piglets as Influenced by Treatments (mean ± s.d.) Treatment week 1 week 2 week 3 week 1 to 3 % of control Feed intake (g/d) Feed A 156 365 472 331 100 Feed B 191 376 536 368 111 Feed C 180 391 533 361 110 Feed D 189 355 469 338 102 Daily growth (g/d) Feed A 127 ± 57  127 ± 57  300 ± 133 185 ± 81  100 Feed B 164 ± 73** 160 ± 70** 301 ± 144 208 ± 95* 112 Feed C 165 ± 90** 161 ± 88** 297 ± 173  207 ± 116* 111 Feed D 141 ± 81** 123 ± 73  280 ± 111 181 ± 71  98 Feed conversion (kg feed/kg growth) Feed A 1.23 2.88 1.57 1.79 100 Feed B 1.16 2.35 1.78 1.77 99 Feed C 1.09 2.43 1.79 1.74 97 Feed D 1.34 2.89 1.68 1.87 104

The visual health score ranged between 4 and 9 on treatment A; while for the other treatments, the range was 8 to 9 without marked differences.

The daily growth did not differ between treatments A and D as well as between B and C. The most pronounced differences were obtained in the first two weeks after weaning during which the best growth performance (with about 30% increase over the control) was obtained with treatments B and C. The better results obtained with the feeds B and C (MCTG2 without or with lipase) are due to an increase of the feed intake. The best feed conversion, however, was obtained with the feed containing MCTG2+lipase. The improvement of the growth using a combination of a MCTG (MCTG2) and a lipase was in the same range as obtained with quinoxalines (additives with both a Gram-positive and Gram-negative spectrum).

Two weeks after weaning, 5 barrows of each experimental group were euthanized. Because the pigs were fed ad libitum, there was no control of the feed intake. Upon dissecting the gastrointestinal tract, samples were taken from the stomach and the upper (duodenum) small intestine, and then analyzed chemically and bacteriologically in the same way as explained in the above experiments. Only the total anaerobic count was reported below.

The pH of the gastric contents was about 3.5 and about 5.7 in the duodenum; there were no differences among the treatments. The total anaerobic counts are reported in Table 7.

TABLE 7 Total Anaerobic Count* in Stomach and Upper Small Intestine of Piglets as Influenced by Different Treatments (n = 5) Treatment Stomach Duodenum A 7.0 ± 0.2 6.4 ± 0.5 B 7.0 ± 0.2 6.1 ± 0.8 C  5.9 ± 0.5**  5.6 ± 0.5** D 6.9 ± 0.2 5.9 ± 0.4 *log₁₀ CFU/g fresh contents, ±s.d.

The results suggest that the feed containing the combination of MCTG2 and L5 caused a significant 10-fold suppression of the bacteriological load, both in the stomach and upper intestine. It is noted that this effect was somewhat lower than in the previous experiment with the gastric cannulated pigs, which could be due to the lower amount of MCTG used in the present experiment (2.5% used herein vs. 5% used previously) and/or the different feeding and sampling procedures. Nevertheless, the present experiment confirmed the results obtained in the cannulated pig reported in above Example 3. The same can be stated for the results of the analysis of the different fat fractions (g/100 g of fresh contents) and the degree of hydrolysis (g FFA/100 g of total FA) in the gastric contents, which are given in Table 8.

TABLE 8 Concentrations* of Free FA and Total FA in Gastric Contents and Degree of Hydrolysis** as Influenced by Treatments in Pigs (n = 5) Treatment A B C D Free FA (FFA) 0.28 ± 0.06 0.44 ± 0.10 0.95 ± 0.22 0.31 ± 0.09 Total FA 1.05 ± 0.08 1.25 ± 0.22 1.35 ± 0.28 1.07 ± 0.17 % hydrolysis 26.7 35.2 70.4 28.9 *g/100 g fresh contents, mean ± s.d. **FFA/total FA in %

It can be calculated from the hydrolysis results that for Feed B (MCTG2) and Feed C (MCTG2+L5), 0.3 and 0.4% free MCFA were present in the stomach, respectively. In the previous experiment with the cannulated pigs, the highest concentrations of free MCFA (and the strongest inhibition of the bacterial load, i.e., about 100 fold) were obtained with MCTG1+L5 and coconut oil+L5, which were 0.60 and 0.30%, respectively.

The combined results of Experiments 2 and 3 clearly suggest that there is a correlation between the amount of released free MCFA and the inhibitory effect on the gastric flora.

Example 4 In Vitro Evaluation of Optimal Combination of Different Concentrations of MCTG with Different Doses of Selected Lipolytic Enzymes

As growth promotion is related and proportional to the inhibition of the total bacterial load in the small intestine, this in vitro experiment was designed to evaluate an optimal combination of different concentrations of MCTG with different doses of lipolytic enzymes, wherein a combination of MCTG1, MCTG2 or coconut oil and a proven effective lipase (L5) was used.

In detail, four concentrations of MCTG were used: 0, 2.5, 5 and 10%; for each of these concentrations, the lipase (L5) was incorporated at 10,000, 1,000 or 100 ppm. The medium contained also 2.5 g per incubation flask of the same synthetic feed (based on starch, dextrose, casein and a vitamin-mineral premix) as used in Experiment 1. However, in the present experiment, the triglyceride was dispersed using gum arabic and gum tragacanth before adding to the medium. The incubations were done at pH 5 using an appropriate acetate buffer. Finally the medium (15 ml) was inoculated with 1 ml of a suspension of bacteria originating from the ileal contents of two cannulated pigs fed a diet without growth promoting additives. Incubations were done for 180 min at 37° C. in a shaking water bath and were done in duplicates.

The methods for the analysis of fats and the bacterial counts were the same as used in the previous experiments with the exception that only the total anaerobic count was reported. As a relationship between the antibacterial activity and the molecular weight of the fatty acid was expected, the results for the free fatty acids were also expressed on a molar basis. The results are given in Table 9.

TABLE 9 Relationship between in vitro Release* of Free Fatty Acids and Total Anaerobic Count** with Different TG and Different doses of L5 Free FA Free FA Total Count (g %) (M) (log₁₀ CFU/ml) MCTG1 Start 0 0 6.2 180 min, control 0 0 6.8 180 min, 10.000 ppm L5 2.5% MCTG1 0.17 0.012 5.9 5% MCTG1 0.34 0.024 <1 10% MCTG1 0.63 0.044 <1 180 min, 1000 ppm L5 2.5% MCTG1 0.11 0.008 6.1 5% MCTG1 0.20 0.014 4.8 10% MCTG1 0.39 0.027 3.8 180 min, 100 ppm L5 2.5% MCTG1 0.09 0.006 6.5 5% MCTG1 0.13 0.009 6.5 10% MCTG1 0.22 0.015 6.2 MCTG2 Start 0 0 6.3 180 min, control 0 0 7.0 180 min, 10.000 ppm L5 2.5% MCTG2 0.17 0.012 5.5 5% MCTG2 0.30 0.021 3.4 10% MCTG2 0.58 0.040 1.8 180 min, 1000 ppm L5 2.5% MCTG2 0.13 0.009 6.3 5% MCTG2 0.21 0.015 6.3 10% MCTG2 0.36 0.025 5.6 180 min, 100 ppm L5 2.5% MCTG2 0.11 0.008 6.5 5% MCTG2 0.16 0.011 6.6 10% MCTG2 0.23 0.016 6.7 COCONUT OIL Start 0 0 6.3 180 min, control 0 0 7.1 180 min, 10.000 ppm L5 2.5% coc. Oil 0.10 0.007 7.2 5% coc. Oil 0.16 0.011 6.2 10% coc. Oil 0.36 0.025 6.2 180 min, 1000 ppm L5 2.5% coc. Oil 0.07 0.005 6.4 5% coc. Oil 0.13 0.009 6.5 10% coc. Oil 0.22 0.015 6.4 180 min, 100 ppm L5 2.5% coc. Oil 0.05 0.003 6.9 5% coc. Oil 0.08 0.006 7.0 10% coc. Oil 0.13 0.009 7.0 *g % or moles (M) in the medium **log₁₀ CFU/ml medium

The above data demonstrate the following results: (1) the amount of released fatty acid is nearly proportional to the concentration of the triglyceride, while a 10-fold dose increase of the lipolytic enzyme used only doubled the concentration of the free fatty acid. For each combination of a specific % of triglyceride and a given ppm of lipolytic enzyme, the release of the fatty acid follows the order: MCTG1>MCTG2>coconut oil; (2) the higher the concentration of the free fatty acid, the more pronounced the suppression of the number of bacteria. A minimal concentration of about 0.35% of fatty acid in the medium appears necessary for a significant suppression of the flora, which concentration corresponds to 0.025 moles/liter. The order of MCTG1>MCTG2>coconut oil corresponds with an increase of the molecular weight of the quantitatively most important MCFA in the triglycerides: MCTG1=C8, MCTG2=C10, coconut oil=C12; and (3) the used in vitro protocol offers an excellent tool for the screening of the numerous combinations of MCTG and available lipolytic enzymes for their usefulness as feed supplements with a stabilizing or suppressive effect on the gastrointestinal microflora. This effect is generally accepted as the basis for obtaining a growth promotion.

TABLE 10 Examples of Commercial Sources ⁽¹⁾ of Chemically Synthesized Structured Lipids Product Composition Company Aldo MCT C8, C10 Lonza Inc., Fair Lawn, USA Stabilox-860 C8, C10 Loders-Croklaan BV, Wormerveer, NL Caprenin C6:0, C8:0, C22:0 Proctor & Gamble, Cincinatti, OH: Salatrim C3:0, C4:0, C18:0 Nabisco Foods Group, East Hanover, NJ Captex C8:0, C10:0, C18:2 Abitec, Columbus, OH Captex 300 C8, C10 Capital City Products, Columbus, OH Captex 810B C8, C18 Capital City Products, Columbus, OH Tripelargonate C9 Capital City Products, Columbus, OH Mixed odd chain C7, C9 Abbott Laboratories, North Chicago, IL Neobee C8:0, C10:0, LCFA Stepan Co, Maywood, NJ Neobee M5 C8:0, C10:0 Stepan Co, Maywood, NJ Neobee 1095 C10:0 Stepan Co, Maywood, NJ Coconado C8:0 Kao Co, Wakayama, Japan Coconard-RK C8, C10, C12 Kao Co, Wakayama, Japan MCTG C4, C5, C6, C7, C8, C10 Karlshamns Lipid Specialties, Columbus, OH MCTG C8, C10 Mead Johnson & Co, Evansville, IN ⁽¹⁾ Source: tested products + literature compilation

TABLE 11 Examples of Experimental or Commercially Available Microbial Lipases ⁽¹⁾ Origin Organism Company Yeast Candida sp. Candida rugosa* Amano, Biocatalysts, Boehringer Mannheim Fluka, Genzyme, Sigma, Meito Sankyo Candida antartica A/B Boehringer Mannheim, Novo Nordisk Candida lipolytica Candida paralipolytica Saccharomyces lipolytica Fungal Thermomyces lanuginosus** Novo Nordisk, Boehringer Mannheim, Amano Rhizomucor Miehei Novo Nordisk, Biocatalysts, Amano Rhizopus sp. Nagase, Tokyo, Japan Rhizopus delemar Rhizopus oryzae Rhizopus niveus Alltech, Rhizopus arrhizus Sigma Rhizopus javanicus Amano Aspergillus sp. Aspergillus niger Finnfeeds International, Amano Aspergillus usamii Aspergillus oryzae Novo Nordisk Mucor sp. Mucor javanicus Mucor lipolyticus Penicillium sp. Penicillium roquefortii Amano Penicillium cyclopium Amano Penicillium simplissimum Penicillium camembertii Geotrichum candidum Amano Neurospora crassa Ustilago maydis Fusarium solani Bacterial Burkholderia cepacia*** Amano, Fluka, Boehringer Mannheim Pseudomonas sp. Pseudomonas alcaligenes Genencor Pseudomonas mendocina Genencor Pseudomonas fluorescens Pseudomonas aeroginosa Amano Pseudomonas spp. Finnfeeds International; Karlan, CA, USA Chromobacterium viscosum**** Asahi, Tokyo, Japan; Biocatalysts; Karlan, CA, USA; Toyo Jozo Shizuoka, Japan Staphylococcus sp. Staphylococcus aureus Staphylococcus carnosus Staphylococcus hyicus Achromobacter lipolyticum Acinetobacter Propionibacterium acnes Bacillus sp. *formerly named Candida cylindracea **formerly named Humicola lanuginosus ***formerly named Pseudomas cepacia ****C. viscosum is identical to Burkholderia glumae ⁽¹⁾ Source: tested products + literature compilation

Example 5 Growth Inhibition of Salmonella Thypimurium Using Compositions Comprising Different Concentrations of MCTG and Lipolytic Enzymes

Salmonella Thypimurium SL 3144 cultures grown in a LB-medium overnight were brought to equal densities (5×10⁸ CFU/ml) and diluted 1.000-fold in 300 μl fresh medium. Bacteria were grown in a 100-well bioscreenplate at 37° C., and the optical density at 600 nm (OD₆₀₀) was measured automatically every 30 min during 6.5 hours in a BioscreenC apparatus. For each time point, the average optical density was calculated from three independent measurements. By plotting the measured optical density versus time, microbial growth curves were generated for the given conditions (pH, MCTG type and concentrations, as well as lipase concentrations). Each point in the growth curve was an average value of the three measurements.

The incubation medium used was LB or M9. The incubation was performed at a temperature correlating well with the animal temperature (37° C.-40° C. for pigs or poultry), and the incubation pH also corresponded to specific pH zones prevailing in the intestinal tract (ranging from 2.5 to 7.0). Test results shown herein were obtained at pH 4.5, 5.2 or 7.0.

Lipase dosage used in the present study ranged from 10 to 80 mg of lipase/g of MCTG. Lipase activity was of 200,000 LU/g lipase. The lipase activity was defined as “Lipase activity Unit” (LU) wherein 1 LU is the amount of enzyme which liberates 1 μmol of fatty acid per minute from olive oil after incubation for 30 minutes at 40° C. and at pH 7.0.

MCTG used was Aveve MCT 8/10 and Aveve MCT 6/8, the compositions of which are shown in Table 12. The concentration of MCTG in the incubation medium ranged from 0.01 to 0.04%.

TABLE 12 FA g/100 g FA Aveve MCT 6/8 C6:0 56.6 C8:0 43.4 Aveve MCT 8/10 C8:0 70.0 C10:0 25.5 C12:0 2.5

The first experiment was performed at pH 4.5 using Aveve MCT 8/10. The concentrations of MCTG and lipolytic enzyme in the test compositions are shown in Table 13. The control (composition H) used in this experiment consisted of incubation medium alone at pH 4.5.

TABLE 13 Composition (pH 4.5) E F G I J K L M N O P Q Aveve MCT 8/10 (%) 0.01 0.02 0.04 0.01 0.02 0.04 0.01 0.02 0.04 0.01 0.02 0.04 Lipase (mg/g of MCTG) 10 10 10 20 20 20 40 40 40 80 80 80

The results are shown in FIGS. 4-7. In detail, FIG. 4 demonstrates the growth inhibition of Salmonella Thypimurium using compositions E, F, G and H; FIG. 5 demonstrates the growth inhibition of Salmonella Thypimurium using compositions I, J, K and H; FIG. 6 demonstrates the growth inhibition of Salmonella Thypimurium using compositions L, M, N and H; and FIG. 7 demonstrates the growth inhibition of Salmonella Thypimurium using compositions O, P, Q and H.

The second experiment was performed at pH 7 using MCT 8/10. The concentrations of MCTG and lipolytic enzyme in the test compositions are shown in Table 14. The control (composition T) used in this experiment consisted of incubation medium alone at pH 7.

TABLE 14 Composition (pH 7) R S Aveve MCT 8/10 (%) 0.02 0.02 Lipase (mg/g of MCTG) 10 40

The results are shown in FIGS. 8 and 9. In detail, FIG. 8 demonstrates the growth inhibition of Salmonella Thypimurium using compositions R and T, whereas FIG. 9 demonstrates the growth inhibition of Salmonella Thypimurium using compositions S and T.

The third experiment was performed at pH 4.5 using Aveve MCT 6/8. The concentrations of MCTG and lipolytic enzyme in the test compositions are shown in Table 15. The control (composition Y) used in this experiment consisted of incubation medium alone at pH 4.5.

TABLE 15 Composition (pH 4.5) U V W X Aveve MCT 6/8 (%) 0.04 0.04 0.04 0.04 Lipase (mg/g of MCT) 10 20 40 80

The results are shown in FIGS. 10-13. In detail, FIG. 10 demonstrates the growth inhibition of Salmonella Thypimurium using compositions U and Y; FIG. 11 demonstrates the growth inhibition of Salmonella Thypimurium using compositions V and Y; FIG. 12 demonstrates the growth inhibition of Salmonella Thypimurium using compositions W and Y; and FIG. 13 demonstrates the growth inhibition of Salmonella Thypimurium using compositions X and Y.

The fourth experiment was performed at pH 5.2 using Aveve MCT 6/8. The concentration of MCTG and lipolytic enzyme in the test compositions are shown in Table 16. The control (composition Y1) used in this experiment consisted of incubation medium alone at pH 5.2. FIG. 14 demonstrates the growth inhibition of Salmonella Thypimurium using compositions V1 and Y1.

TABLE 16 Composition (pH 5.2) V1 Aveve MCT 6/8 (%) 0.04 Lipase (mg/g of MCT) 20

CONCLUSIONS

All MCTG/lipase incubations with different MCTG types and different concentrations of MCTG and lipase showed inhibition of the growth curve of the bacterium tested. The data shows that the inhibition of bacterial growth is detectable starting from a concentration of 0.01% MCTG and 10 mg lipase/g of MCTG. Growth curve reduction in the time frame of 0-6.5 h correspond to a normal digestion time in pigs and is longer than the digestion time in poultry.

The results presented herein clearly show reduced growth of the bacterium tested at different pH ranges which was dependent of the concentration of MCTG in the incubation medium. Activity of the lipase at a concentration of 10-80 mg/g of MCTG induced a reduction of the microbial growth. The above lipase concentration in the incubation medium with 0.01%-0.04% MCTG corresponds to 1 ppm to 32 ppm of lipase in the incubation medium.

The antimicrobial activity demonstrated in the present study is consistent with the in vivo bacterial reduction measurement, which is from 30% to 100% reduction of bacterial counts depending on the bacterial group tested.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

-   1. Bach, A. C. & Babayan, V. K., 1982, Medium-chain Triglycerides:     an Update, The American Journal of Clinical Nutrition, 36: 950-962. -   2. Cera, K. R. et al., 1989, Postweaning Swine Performance and Serum     Profile Responses to Supplemented Medium-chain Free Fatty Acids and     Tallow, Journal of Animal Science, 67, 2048-2055. -   3. Chesson, A., 1994, Probiotics and other Intestinal Mediators, In:     Principles of Pig Science, D. J. A. Cole, J. Wiseman & M. A. Varley,     Editors, Nottingham University Press, UK, pp. 197-214. -   4. Corpet, D. E., 1996, Microbial Hazards for Humans of     Antimicrobial Growth Promotor Use in Animal Production, Revue     Medicine Vétérinaire, 147: 851-862. -   5. Decuypere, J. A. et al., 1977, Gastro-intestinal Cannulation in     Pigs: a Simple Technique allowing multiple Replacements, Journal of     Animal Science, 46, 463-468. -   6. Kabara, J. J., 1978, Fatty Acids and Derivates as Antimicrobial     Agents—a Review, In: The Pharmacological Effects of Lipids, J. J.     Kabara, Editor, The American Oil Chemists Association, Champaign,     II, USA, pp. 1-14. -   7. Odle, J., 1999, Medium-chain Triglycerides: a Unique Energy     Source for Neonatal Pigs, Pig News and Information, 20: 25N-32N. -   8. Lepine, A. J. et al., 1989, Effect of Colostrum or Medium-chain     Triglyceride Supplementation on the Pattern of Plasma Glucose,     Non-esterified Fatty Acids and Survival of Neonatal Pigs, Journal of     Animal Science, 67, 983-990. -   9. Moreau, H. et al., 1988, Screening of Preduodenal Lipases in     several Mammals, Biochemica Et Biophysica Acta, 959, 247-252. -   10. Partridge, G. G., 1993, New Approaches with Pig Weaner Diets,     In: Recent Advances in Animal Nutrition, P. C. Gansworthy & Cole, J.     A., Editors., Nottingham University Press, UK, pp. 221-248. -   11. Roth, F. X. & Kirchgessner, 1998, Organic Acids as Feed     Additives for young Pigs: Nutritional and Gastrointestinal Effects,     Journal of Animal and Feed Sciences, 7: 23-33. -   12. SPSS for WINDOWS, 1997, User's Guide (Release 7.5), SPSS Inc.,     Chicago, Ill. 60611. -   13. Van der Heyde, H. & Henderickx, H., 1963, Zur Vereinfachung der     quantitativen und qualitativen Bestimmung der Bakterien unter     Verwendung von “Ringplatten”, Zentralblatt Für Bakteriologie, I     Orig., 189, 224-228. -   14. Van der Peet, G. F. V., 1992, Voeding van jonge Biggen,     CVB-Documentatierapport N^(o)5. -   15. Wegener, H. C. et al., 1998, The Association between the Use of     Antimicrobial Growth Promoters and Development of Resistance in     Pathogenic Bacteria towards Growth Promoting and Therapeutic     Antimicrobials, Journal of Animal and Feed Science, 7: 7-14. 

1. A feed composition comprising about 0.01% to about 20% by weight of triglycerides and about 1 ppm to about 10,000 ppm of exogenous active lipolytic enzyme, wherein said triglycerides contain about 50% to about 100% of C₆-C₁₀ medium chain fatty acids (MCFA).
 2. The feed composition according to claim 1, wherein said triglycerides contain about 60% to about 100% of C₆-C₁₀ MCFA.
 3. The feed composition according to claim 2, wherein said triglycerides contain about 70% to about 100% of C₆-C₁₀ MCFA.
 4. The feed composition according to claim 3, wherein said triglycerides contain about 80% to about 100% of C₆-C₁₀ MCFA.
 5. The feed composition according to claim 4, wherein said triglycerides contain about 90% to about 100% of C₆-C₁₀ MCFA.
 6. The feed composition according to claim 5, wherein said triglycerides contain about 100% of C₆-C₁₀ MCFA.
 7. The feed composition according to any one of claims 1 to 6, wherein said triglycerides are selected from caproic/caprylic/capric triglycerides, heptanoic/pelargonic triglycerides, pelargonic triglycerides, heptanoic triglycerides, caprylic/capric triglycerides, caproic/caprylic triglycerides, caproic/capric triglycerides, caproic triglycerides, caprylic triglycerides, capric triglycerides, and combination thereof.
 8. The feed composition according to claim 7, wherein said triglycerides are selected from caproic/caprylic/capric triglycerides, caprylic/capric triglycerides, caproic/caprylic triglycerides, caproic/capric triglycerides, caproic triglycerides, caprylic triglycerides, capric triglycerides, and combination thereof.
 9. The feed composition according to claim 1, wherein said exogenous active lipolytic enzyme is a lipase.
 10. The feed composition according to claim 1, wherein said exogenous active lipolytic enzyme is an esterase.
 11. The feed composition according to claim 1, wherein said exogenous active lipolytic enzyme is a mixture of lipase and esterase.
 12. The feed composition according to claim 1, further comprising an animal feed, wherein said animal feed comprises carbohydrate, protein, saccharides and a vitamin-mineral premix.
 13. The feed composition according to claim 12, wherein said animal feed comprises starch, dextrose, casein and a vitamin-mineral premix.
 14. The feed composition according to claim 1, further comprising an animal feed, wherein said animal feed comprises maize, barley, wheat, dried acid whey, cassaya, herring meal, soybean, soy-flour and a vitamin-mineral premix.
 15. The feed composition according to claim 1 for feeding early weaned animals.
 16. The feed composition according to claim 15, wherein said animal is piglet.
 17. The feed composition according to claim 1 for use as an antimicrobial agent.
 18. The feed composition according to claim 1 for preventing digestive upsets.
 19. The feed composition according to claim 1 for use as a growth promoter.
 20. A method for controlling lipolysis in the gastro-intestinal tract of an animal, comprising the step of administering to said animal a therapeutically effective amount of the feed composition according to claim
 1. 21. The method according to claim 20, wherein said animal is an early weaned piglet.
 22. A method for treating growth impairment of an animal, comprising the step of administering to said animal a therapeutically effective amount of the feed composition according to claim
 1. 23. The method according to claim 22, wherein said animal is an early weaned piglet.
 24. A method for treating digestive upsets of an animal, comprising the step of administering to said animal a therapeutically effective amount of the feed composition according to claim
 1. 25. The method according to claim 24, wherein said animal is an early weaned piglet. 